Tissue Engineering of Cartilage

from Human Adipose Tissue

Wan Kee Min

The Graduate School

Yonsei University

Department of Medicine

- ii -

Tissue Engineering of Cartilage

from Human Adipose Tissue

A thesis

Submitted to the Department of Medicine

and the Graduate School of Yonsei University

in partial fulfillment of the

requirements for the degree of

Doctor of Medicine

Wan Kee Min

June 2005

- iii -

This certifies that the thesis of

Wan Kee Min is approved

__________________________________

Thesis Supervisor: Yoon Kyu Chung

__________________________________

Joo Young Park: Thesis Committee Member #1

__________________________________

Won Soo Lee : Thesis Committee Member #2

__________________________________

Dong Joon Park : Thesis Committee Member #3

__________________________________

Kyoung Ho Lee : Thesis Committee Member #4

The Graduate School

Yonsei University

June 2005

- iv -

감감감사사사의의의 글글글

감사합니다.초여름 싱그러운 녹색이 산천에 물드는 아름다운 계절입니다.

여기 이 자리에 오기까지 여러 면에서 도와주시고 이끌어주신 정윤규 선생님,개인적으로도 앞으로 살아가야할 제 삶의 방향을 몸소 보여주시고계셔서 항상 존경과 흠모의 염을 가지고 살고 있습니다.막바지에 미국에서 연구와 강의를 하고 있는 성도유 선생님은 논문 작

성과 교정에 열 일 마다하고 무한한 도움을 주셨습니다.이 자리를 빌려 도움을 주신 여러분들의 관심과 애정에 감사드립니다.

개업해서 많은 어려움을 겪으면서도 학위논문을 준비할 수 있었던 것은가족들의 믿음과 성원이 없었다면 견디기 힘든 나날들이었을 겁니다.앞으로도 이런 여러분들의 기대와 성원을 저버리지 않도록 열심히 성실히살겠습니다.

민 완 기 올림

- v -

Contents

Abstract ··············································································································· viii

Introduction ············································································································ 1

Materials and Methods ························································································· 3

Fat harvest ··································································································· 3

Extractoin of stem cells ············································································· 3

Chondrogenesis ···························································································· 4

Analysis to confirm chondrogenesis ························································· 4

Results ···················································································································· 7

Discussion ·············································································································· 8

Reference ·············································································································· 11

Abstract in Korean ····························································································· 14

Figure ··················································································································· 16

‧

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Contents

Figure and Table

Table 1.

Primer sequences of gene products for RT-PCR. ···································· 6

Fig-1.

Processed human fat tissue into putative stem cell. 2-D (x100) ········· 16

Fig-2.

Plated PLA transforming into chondrocytes in chondrogenesis medium.

(x100) after 1 day (left) and 6 days. 2-D (right) ································· 16

Fig-3.

Photographs showing in vitro chondrogenic induction of fat cells

resulting in chondrocyte formation. 3-D A: H&E x200, B: H&E x400

........................................................................................................................17

- vii -

Fig-4.

Chondrogenic induction of fat cells confirmed by Alcian Blue at pH

1.0. 3-D A: x40, B: x100 ········································································· 18

Fig-5.

PLA derived chondrocytes shown at 6 weeks. Note in vitro

chondrogenic induction of PLA cells resulting in a transitory

fibrohyaline cartilage. 3-D (H&E, x200(A), x400(B))··························· 19

Fig-6.

Alcian Blue staining at 6 weeks. 3-D (x100(A), x400 (B)) ················ 20

Fig-7.

RT-PCR analysis of cartilage at 6 weeks. ·············································· 21

- viii -

Abstract

The fact that cartilage has little self-healing potential reflects the reason for

vast research in this field. Recent evidence indicates that processed

lipoaspirates (PLA) in adipose tissue may be a source of chondrocytes which

produce matrix proteins of cartilage. The present study was to confirm the

above observation and to investigate the feasibility of fibrin glues as a scaffold

for cartilage tissue engineering approach. The results showed that the cells

differentiated from PLA in fibrin glue were positive for proteoglycan, cartilage

matrix protein, using Alcian Blue staining technique. Moreover, these cells also

expressed type II and X collagen, aggrecan, and phenotypes of chondrocytes

by reverse transcriptase-polymerase chain reaction technique. The findings of

this study indicate that fibrin glue may be a successful carrier of chondrocytes

in well-defined nodules from PLA.

- 1 -

Tissue Engineering of Cartilage

from Human Adipose Tissue

<Directed by professor Yoon Kyu Chung >

The Graduate School

Yonsei University

Department of Medicine

Wan Kee Min

Introduction

Cartilage, a combination with chondrocytes and extracellular matrix, is

widely distributed in the human body. Unfortunately, the remodeling rate of

cartilage is so slow that the damaged cartilage has been shown to have a

limited potential of repair. Eventually, the extended injury area of cartilage to

the subchondrol bone is substituted by fibrocartilage, which has inferior quality

to the original cartilage. Thus, it is necessary to explore a promising method

for the repair of cartilaginous defects.

Within decade, cartilage tissue engineering is most like to benefit from a

source of autologous multipotent stem cells such as bone marrow stroma.

- 2 -

Among the many cells in the bone marrow, mesenchymal stem cells (MSCs)

are capable of differentiating into chondrogenic cells. However, the clinical use

of mesenchymal stem cells (MSCs) has several obstacles, including pain and

incontinence in aspiration of bone marrow from spine and low cell yield upon

harvest. Recently, the human adipose tissue has been reported to have

multi-lineage cells (processed lipoaspirate, PLA).1-5 The adipose tissue can be

significant as a source for mesenchymal stem cells due to its volume and

accessibility.

Furthermore, if the tissue-engineered cartilage can be produced in an

injectable form, minimal invasive chondrocyte implantation would be possible.

The ideal, injectable, tissue-engineering polymer should be non-reactive,

maintain a three-dimensional configuration, and degrade at the appropriate rate.

Fibrin glue, a non-reactive and rapidly degrading biologic polymer, offers

several advantages over various polymers. By using a thrombin glue, which has

decreased concentration of the thrombin component on the fibrin glue, the

fibrin substrate remains liquid enough to allow percutaneous injection of the

polymer. As the glue polymerizes, it should mold into any form regarding its

surrounding structure. It has been shown that fibrin-based polymers produce

high quality neocartilage when seeded with chondrocytes,6 This would be ideal

for the neocartilage to be delivered in a minimal invasive fashion.

This paper examined the chondrogenic potential of multipotential cells from

human adipose tissue and to develop a tissue-engineered cartilage using fibrin

glue.

- 3 -

Materials and Methods

Fat harvest:

After informed consent and approval, fat was harvested from transverse

rectus abdominis flap, abdomen liposuction, or abdominoplasty procedures.

Extraction of stem cells:

The raw fat was washed extensively with sterile phosphate buffered saline

(PBS) to remove blood cells, saline, and local anesthetics. The extracellular

matrix was digested with 0.07% collagenase (Sigma. St. Louis, MO, USA) at

37°C for 30 minutes to release the cellular fraction. And then collagenase was

inactivated with an equal volume of Dulbecco Modified Eagle Medium

(DMEM: GibCo, Carlsbad, CA, USA) containing 10% fetal bovine serum

(FBS: GibCo, Carlsbad, CA, USA). The infranatant was centrifuged at 250 g

for 10 minutes to obtain a high density PLA. The pellet was resuspended in

DMEM and 10% FBS and then mixed with an erythrocytes lysis buffer (0.16M

NH4Cl) for 10 minutes to lyse contaminating erythrocytes. After an additional

centrifugation step, PLA was resuspended in DMEM and 10% FBS and plated

in 100mm tissue culture dishes at a density of 1×107 cells per plate. PLA was

maintained in control medium (control medium was composed of Dulbecco

Modified Eagle Medium, 10% fatal bovine serum, 1% antibiotic/antimycotic).

- 4 -

The culture medium was changed twice weekly..Confluent PLA (approximately

80% confluence) was passaged at a ratio of 1:3 in trypsin/ethylenediaminetetra

-acetic acid (GibCo, Carlsbad, CA, USA).

Chondrogenesis:

1×105 PLA was plated into the center of each well (a 6 well plate) and

allowed to attach at 37°C for 2 hours. PLA was maintained in chondrogenic

medium (CM) consisting of DMEM, 10% FBS, 100 ng/ml of insulin (Sigma,

St. Louis, USA), 50 ug/ml of ascorbic acid-2-Phosphate (Sigma, St. Louis,

USA ), and 5 ng/ml transforming growth factor β-1(Biosource, Carmarilo, CA,

USA) for a week. After one week, cells differentiated from PLA was harvested

with collagenase and collected by centrifugation at 250 g for 10 minutes. The

supernatant was removed and the cell pellet was added with 10 ml of CM to

make a concentration of 1×106 cells/ml. The cells in 10 ml of CM were

overlaid on prepared fibrin glue (80 mg/cc, a mixture of 0.25 cc of thrombin

and 0.25 cc of fibrinogen, Baxter, Deerfield, IL, USA) in 50 ml of tube and

then were centrifuged to allow the cells to enter fibrin glue. Entrapped cells

were centrifuged everyday and incubated with CM at 37°C with 5% CO₂ for

2 or 6 weeks for the analyses.

Analysis to confirm chondrogenesis:

-Staining

.At 2 and 6 weeks of incubation period, fibrin glue containing cells were

- 5 -

fixed with 4% paraformaldehyde for 15 minutes at room temperature, washed

with several changes of PBS, embedded in paraffin, and cut with the size of

6μm. Sliced paraffin were mounted on slides and stained using standard

hematoxylin and eosin (H&E) according to the instruction of protocol or

incubated with 1% (wt./vol.) Alcian Blue (Sigma, St. Louis, MO, USA) in

01.N HCl (pH 1.0) for 30 minutes. Digital images were obtained using a Zeiss

Axioskop II microscope and Spot software.

-Extraction of RNA/gene expression by

reverse transcriptase-polymerase chain reaction (RT-PCR)

At 6 weeks of incubation period, total RNA was isolated from PLA ...and

differentiated chondrocytes from PLA in fibrin glue using Trizol

.(Invitrogen, St. Louis, USA). Isolated total RNA was denatured at 70°C for

5 minutes and cooled on ice. And then incubated with dNTP and MMLV-RT

(Timesaver cDNA .synthesis kit, Amersham biosciences, Piscataway, NJ, USA)

for a minimum of 2 hours at .42°C. For PCR amplification of the cDNA, used

primer pairs designed to .the following genes were presented in Table 1

(F=forward oligo,. R=reverse oligo). cDNAs using PCR were amplified by

95°C for 1 minute, 53°C for 1 minute, 72°C for 1 minute with 35 cycles and

then the PCR products were extended at 72°C for 5.minutes for the end of

reaction. .PCR using primers to ß-actin was used as a positive control. PCR

products were resolved using convectional agarose gel electrophoresis.

- 6 -

Table 1. Primer sequences of gene products for RT-PCR

Gene Sequence Size(bp)

ß-actin F .5'-TGTGATGGRGGGAATGGGTCAG-3' 360 bp

. R 5'-TTTGATGTCACGCACGATTT-3'

Type ll collagen αααα1 F 5'-CCAAGTACTTTCCAATCTCAGTCAC-3' 328 bp

R 5'-ACAGAAAGCACCATTGTGTAGGAC-3'

Aggrecan .F 5'-GCGATATCATGACCACTTTACTC-3' 500 bp

R 5'-CCTGTCAAAGTCGAGGGTGT-3'

Type X collagen F 5'-TGGAGTGGGAAAAAGAGGTG-3' 600 bp

R 5'-GTCCTCCAACTCCAGGATCA-3'

- 7 -

Results

This study examined the chondrocyte lineage potential of mesenchymal stem

cell population obtained from human fat tissue. Raw human fat tissues were

used to obtain stem cell fraction, PLA (Fig-1). The PLA was cultured in CM

at 1 day and 6 days (Fig-2) The nodules stained with H&E contained

chondrogenic cells within lacunae surrounded by abundant gel-like extracellular

matrix at 2 weeks (Fig-3). At 2 weeks, nodules also positively responded with

the staining of Alcian Blue, an indicator of proteoglycan which is a cartilage

matrix protein (Fig-4). At 6 weeks, a enhanced definition of PLA derived

cartilages are shown using H&E and Alcian Blue staining (Fig-5 and 6). The

RT-PCR analysis confirmed the presence of phenotype of chondrocytes, e.g.,

type II collagen and aggrecan in all nodules (Fig-7). Type X collagen, which

is an indicator of progression change into hypertrophic chondrocytes was also

present (Fig-7). On the other hand, PLA did not express any phenotype of

chondrocytes (Fig-7).

- 8 -

Discussion

The results in this paper confirm chondrocyte formation from adult stem

cells in fat tissue. Also this study shows the possibility of successfully

producing a tissue engineered cartilage within liquid polymers. The healing of

articular cartilage defects with bone marrow derived stem cells has been

successfully demonstrated in small animal models.7-9 However, the use of bone

marrow may have several disadvantages compared to that of fat tissue for stem

cells. Harvesting stem cells from fat tissue reduces pain and morbidity of the

donor site associated with harvesting of autogenous chondrocytes or bone

marrow. The abundant number of cells in fat tissue eliminates a second stage

procedure for reimplantation of the induced cells. Previous studies have shown

that a bone marrow aspirate of 30ml produces approximately 1×105 cells,

while a liposuction aspirate yields approximately 3.3×104 cells/mm³ of fat.10

Furthermore, the fat cells have high differentiation capacity, leading to

relatively easily transferred to chondrocytes. Nonetheless, further laboratory and

clinical work would be needed to evaluate the efficacy and density of the

actual cartilage formed.

Cartilage is composed of a mixture of collagen fibrils and proteoglycans,

which has the high tensile strength.¹¹﹒¹² Alcian Blue staining confirmed the

presence of proteoglycan within the processed lipoaspirates in the first two

weeks. The expression of type II collagen in RT-PCR analysis is highly

suggestive of cartilage produced in a nonchondrogenic cell type origin. The

- 9 -

expression of type II collagen in processed lipoaspirate cells is consistent with

its detection in embryonic tissue and precartilage mesenchymal precursors

before chondrogenic differentiation. Aggrecan, as shown in the analysis, has

been also demonstrated to accumulate at the onset of overt chondrogenesis,

coincident with cellular condensation. Taken together with these expression, it

is well indicative of chondrogenic capacity of processed lipoaspirate.

The initial studies on the formation of neocartilage have been reported that

the possible use of solid polymers such as polyglycolic acid polymer with

chondrocytes was necessary to surgically place the solid polymer/chondrocyte

into the nude mouse in the purpose of neocartilage construction. With a recent

tissue application, this would reveal additional scars from surgical incisions.

Recent advanced tissue-engineering techniques have led to the use of liquid

form of biodegradable polymer, fibrin glue in the formation of neocartilage. By

using polymers in liquid form, it will become possible to inject the mixture of

chondrocytes and fibrin glue into various parts of the body and then to mold

the preexisting anatomical form. Previous study demonstrated that

subcutaneously implanted fibrin glue with articular or non-articular

chondrocytes into nude mice resulted in the formation of cartilages. The

finding of the present study for the first time shows that differentiated

chondrocytes from PLA in adipose tissue are also able to form neocartilage in

liquid form of fibrin glue. Further studies in an in vivo model will solidify its

efficacy.

In conclusion, chondrogenic capacity of the adult fat cells was confirmed

- 10 -

using a liquid based polymer. Further study will reveal its clinical efficacy.

- 11 -

Reference

1. Znk P.A., Zhu M., Mizuno H., Huang J., Futrell J.W., Katz A.J., et

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2. Lucas P.A., Calcutt A.F., Ossi P., Young H.E., Southerland S.S. Mese

-nchymal stem cells from granulation tissue. J. Cell Biochem. 17E:122,

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3. Young H.E., Mancini M.L., Wright R.P., Smith J.C., Black Jr. A.C.,

.Heagan C.R., et al. Mesenchymal stem cells reside within the connec

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4 .Dragoo J.L., Samimi B., Zhu M., Hame L., Thomas B.J., Lieberman

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5. Hedrick M.H., Daniels E.J. The use of adult stem cells in regenerative

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using plasma derived polymer substrates and chondrocytes. Plast. Reconstr.

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7. Butnariu-Ephrat M., Robinson D., Mendes D.G., Halperin Nevo Z. Resur

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.marrow. Clin. Orthop. 330:234, 1996

- 12 -

8. Angele P., Kujat R., Nerlich M., Yoo J., Goldberg V., Johnstone B. Engineering

of osteochondral tissue with bone marrow mesenchymal progenitor cells in

a devitalized hyalurona-gelatin composite sponge. Tissue eng. 5:545, 1999

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of large, full thickess defects of articular cartilage. J. Bone Joint Surg.

(Am). 76:579, 1994

10. Bruder S.P., Jaiswal N., Haynesworth S.E. Growth kinetics, self-renewal,

and the osteogenic potential of purified human mesenchymal stem cell

during extensive subcultivation and following cryopreservation. J. Cell

Biochem. 64:278, 1997

11. Kosher R.A., Kilyk W.M., Gay S.W. Collagen gene expression during limb

.cartilage differentiation. J. Del. Biol. 102:1151, 1986

12. Lisenmayer T.F., Eavey R.D., Schmid T.M. Type X collagen: A hypertro

-phic cartilage specific molecule. Pathol. Immunopat. Res. 7:14, 1998

13. Dessau W., Von der Mark H., Von der Mark K., Fischer S. Changes in

pattern of collagens and fibronectin during limb bud chondrogenesis. J.

Embryol. Exp. Morphol. 57:51, 1980

14. Linsenmayer T.F., Toole B.P., Trelstad R.L. Temporal and spatial

transition in collagen types during embryonic chick limb devel

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15. Poliard A., Nifuji A., Lambin D., Plee E., Forest C., Kellerman O. Cont

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osteogenic, chondrogenic, or adipogenic pathways. J. Cell Biol. 130:1461,

- 13 -

1995

16. Kosher R.A., Solursh M. Widespread distribution of type II collagen

.during embryonic chick development. Del. Biol. 131:558 ,1989

17. .Cheah K.S., Lau E.T., Au P.K., Tam P.P. Expression of the mouse α-1

.(II) collagen is not restricted to cartilage during development. Develop

.-ment. 111:945,1991

18. ..Kosher R.A., Gay S.W., Kamanitz J.R., et al. Cartilage proteoglycan

.core protein gene expression during limb cartilage differentiation. Dev.

.Biol. 118:112, 1986

19. Johnson T.S., XU J.W., Zaporojan V.V., Mesa J.M., Weinand C.,

.Randolph M.A., Bonassar L.J., Winograd J.M., Yaremchuk M.J. Integ

-rative repair .of .cartilage with articular and nonarticular chondrocytes.

.Tissue Eng. 10.:1308, 2004

- 14 -

국국국문문문요요요약약약

인인인간간간 지지지방방방 체체체세세세포포포에에에서서서 연연연골골골조조조직직직배배배양양양

<지도 교수 정정정 윤윤윤 규규규 >

연세대학교 대학원 의학과

민민민 완완완 기기기

연골이 자기 회복 가능성을 적게 가지고 있다는 사실은 이 분야에서 많은 연구가 이루어져야 하는 이유를 반영한다.최근 논문들에서 인간 지방체 세포의 processedlipoaspirates(PLA)가 연골의 matrixprotein을 생성하는 연골 세포의 근원일 가능성들을 시사하고 있다.이 연구는 위 가능성을 확인,증명하기 위한 것이고 fibringlue가 연골 조직 배양의 scaffold가될 수 있다는 가능성을 연구한 것이다.결과는 fibringlue에 있는 PLA에서 분화된 세포가 proteoglycan,cartilagematrixprotein을 생성한다는 것을 AlcianBluestaining을 통해 확인할 수 있었다.이 세포는 또한 reversetranscriptase-polymerasechainreactiontechnique에 의하여 typeII,Xcollagen,aggrecan,chondrocytes의 표현형을 발현했다.이 연구 결과는fibringlue가 PLA에서 well-definednodules에 있는 chondrocytes의 성공적인 carrier일 수 있다는 것을 보여준다.

- 15 -

Keyword:연골 조직배양,인간 지방 체 세포

- 16 -

Figure-1.

Processed human fat tissue into putative stem cell. 2-D (×100)

Figure-2.

Plated PLA transforming into chondrocytes in chondrogenesis medium (×100)

after 1 day (left) and 6 days. 2-D (Right)

- 17 -

Figure-3.

Photographs showing in vitro chondrogenic induction of fat cells resulting in

chondrocyte formation. 3-D

A: H&E ×200

B: H&E ×400

- 18 -

Figure-4.

Chondrogenic induction of fat cells confirmed by Alcain Blue at pH 1.0. 3-D

A: ×40

B: ×100

- 19 -

Figure-5.

PLA derived chondrocytes shown at 6 weeks. Note in vitro chondrogenic

induction of PLA cells resulting in a transitory fibrohyaline cartilage. 3-D

(H&E, ×200(A), ×400(B))

- 20 -

Figure-6.

Alcian Blue staining at 6 weeks. 3-D (×100(A), ×400(B))

- 21 -

Figure-7.

RT-PCR analysis of cartilage at 6 weeks.

1. β-actin

2. Human type Ⅱ collagen α 1

3. Aggercan

4. Human type X collagen